1. Field of the Invention
The present invention relates to an enzyme electrode. More particularly, the present invention is concerned with an enzyme electrode comprising: (a) an electrode system comprising an insulating substrate having formed thereon a working electrode, a counter electrode and optionally a reference electrode; and (b) a reaction layer formed on the electrode system, wherein the reaction layer comprises diaphorase (DI), 12α-hydroxysteroid dehydrogenase (12α-HSD) and nicotinamide adenine dinucleotide synthetase (NADS), at least a portion of the reaction layer being superposed on the working electrode, wherein the DI, 12α-HSD and NADS contained in the above-mentioned portion of the reaction layer are immobilized on the surface of the working electrode, so that a compound generated in the reaction layer can reach the surface of the working electrode.
In addition, the present invention is concerned with a method for determining the concentration of adenosine triphosphate (ATP) by using the above-mentioned enzyme electrode, and also concerned with a system, which comprises the above-mentioned enzyme electrode, for determining the concentration of ATP.
The enzyme electrode of the present invention is advantageous for miniaturizing an apparatus for determining the concentration of ATP. Further, by the use of the enzyme electrode of the present invention, it has become possible to perform determination of the ATP concentration of a sample easily and rapidly, with high sensitivity, without the need of a cumbersome pretreatment.
2. Prior Art
Adenosine triphosphate (ATP) is a compound which is almost ubiquitously present in many living organisms.
Many chemical reactions occurring in a living body are performed utilizing the energy which is released during the generation of adenosine diphosphate (ADP) or adenosine monophosphate (AMP) by the hydrolysis of ATP. Further, in a living body, ATP is used as a precursor for a ribonucleic acid (RNA) and as a phosphate donor for phosphorylation in vivo.
Accordingly, ATP is a compound which plays a very important role in a living body and, thus, a quantitative analysis of ATP is important in various fields.
For example, in the field of food hygiene and sanitation, the ATP concentration is used as an index of the degree of contamination with microorganisms (such as bacteria) or with remnants of food which are likely to be causative of microbial contamination.
When microorganisms or food remnants are attached to foods or equipments, such as tablewares, kitchen utensils and food processing machines, the amount of ATP detected from the surface of the foods or equipments becomes increased by ATP derived from the microorganisms or food remnants. Therefore, samples obtained from the surface of the foods or equipments are subjected to a quantitative analysis of ATP to thereby determine the degree of contamination. It is considered that the higher the ATP concentration of a sample, the higher the degree of contamination with the microorganisms or with food remnants.
Since the amount of ATP in a sample is very small, a highly sensitive method for accurately determining the ATP concentration is necessary for determining the degree of contamination. As a method for determining the degree of contamination, there is known a method which uses the luciferin-luciferase reaction for determining the ATP concentration. This method involves the steps of reacting luciferin and luciferase with ATP extracted from a sample to thereby cause luminescence, and determining the degree of contamination of the sample from the intensity of luminescence.
In this method, the degree of contamination is determined using a calibration curve which shows the correlation between the intensity of luminescence and the degree of contamination, wherein the calibration curve is prepared by conducting the above-mentioned steps with respect to several standard samples having predetermined degrees of contamination.
The method based on the luciferin-luciferase reaction is performed using an expensive apparatus which is generally large and, hence, difficult to move. Accordingly, the above-mentioned method cannot be suitably used in the analyses in which the transfer of the apparatus is necessary. For example, the above-mentioned method is inapplicable to field analyses in which an analytical apparatus needs to be moved to the outdoors or the place to be protected from microbial contamination so that a desired analysis can be performed there.
Further, in this method, ATP is detected by using an optical technique and, consequently, there is a drawback in that an electric power consumption of the apparatus used for the analysis is large.
In addition, it is generally difficult to analyze a turbid sample by such an analytical method using an optical technique. Therefore, the above-mentioned method is inappropriate for analyzing a highly turbid sample, e.g. milk or blood, as such and, before the analysis of such a turbid sample, there is a need to dilute the sample or subject the sample to a pretreatment for removing or dissolving the insoluble microparticles which are causative of the turbidity. As a result, this method is accompanied by problems, such as cumbersomeness and low sensitivity.
In this situation, an enzyme electrode for quantitative analysis of ATP is receiving attention as an apparatus which not only can be easily moved, but also can be operated with a small electric power consumption and which is compact and is capable of directly analyzing highly turbid samples.
An enzyme electrode is a type of a so-called biosensor, and it is a device in which a change in the amount of a substance caused by an enzyme reaction is converted into electric signals by an electrochemical technique. Detection and measurement of a specific substance can be conducted using the electric signals.
Methods for determining the concentration of a specific substance in a sample by using an enzyme electrode are well known in the art. These methods are recently receiving attention due to their advantageous features, such as rapidness, easiness and economical advantages, and the use of enzyme electrodes in the field of clinical examination and the like are beginning to expand.
Unlike the analysis based on an optical technique, analysis using an enzyme electrode is advantageous in that even a turbid sample as such can be analyzed without the need of dilution thereof and the like and, hence, the analysis can be performed with very simple operations.
An example of an enzyme electrode well known in the art is an enzyme electrode for determining the glucose concentration of a blood sample (see Japanese patent No. 2517153). This enzyme electrode comprises: an electrode system comprising an insulating substrate having formed thereon a working electrode and a counter electrode, wherein the electrodes are prepared by screen printing and the like; an insulating layer formed on the electrode system (the insulating layer is used to maintain the surface areas of the exposed portions of the electrodes at a predetermined value); and an enzyme reaction layer comprising a hydrophilic polymer, an oxidoreductase and an electron carrier which is formed on the electrode system.
As an enzyme electrode for the quantitative analysis of ATP, an enzyme electrode employing glucose oxidase and hexokinase has been proposed (see Unexamined Japanese Patent Application Laid-Open Specification No. Sho 60-17347 (corresponding to U.S. Pat. Nos. 4,711,245 and 4,758,323) and Analytica Chimica Acta, 1997, vol. 340, pp. 109–113).
Glucose oxidase promotes the oxidation of glucose, and hexokinase employs ATP as a phosphate donor to promote the phosphorylation of glucose. Since both of the enzymes use glucose as their substrate, the above-mentioned two enzyme reactions (former and latter reactions) proceed simultaneously when the enzyme electrode is used for analyzing ATP in a sample, and the number of the latter reactions occurring in the enzyme electrode increases in accordance with the increase in the amount of ATP in the sample. For analyzing ATP, the above-mentioned enzyme electrode utilizes this phenomenon.
As another type of enzyme electrode for the quantitative analysis of ATP, there is known an enzyme electrode which uses adenosine triphoshatase (ATPase) and a hydrogen ion-sensitive field-effect transistor (pH-ISFET) (see Unexamined Japanese Patent Application Laid-Open Specification Nos. Sho 61-122560 and Sho 61-269058).
A pH-ISFET is a semiconductor element having a function to convert the change in the hydrogen ion concentration into electric signals.
Hydrogen ions are generated during the hydrolysis of ATP by ATPase, and this leads to an increase in the hydrogen ion concentration. The above-mentioned enzyme electrode is a device which converts such increase in the hydrogen ion concentration into electric signals by means of pH-ISFET, and the electric signals are used to determine the amount of ATP.
With respect to the enzyme electrodes mentioned above, the measuring sensitivity to ATP was only about 10−4 M to 10−5 M (see page 332 of “Fukyuban Sensa Gijyutu (Sensor Technology, Popularized version)” published by Fuji Technosystem Co. Ltd., Japan (1976)), and such enzyme electrodes were inappropriate for practical use.
As an example of enzyme electrodes which have sufficient sensitivity for use in the quantitative analysis of ATP, there can be mentioned an enzyme electrode which uses pyruvate kinase, hexokinase and glucose-6-phosphate dehydrogenase (Electroanalysis, 1991, vol. 3, pp. 659–663).
In this enzyme electrode, pyruvate kinase and hexokinase are used to conduct an enzymatic cycling of ATP/ADP system so as to enhance the sensitivity of the enzyme electrode. An enzymatic cycling is a method for enhancing the sensitivity of a measurement in which a change in the amount of a substance is amplified by using a combination of two or more enzymes.
In the above-mentioned enzyme electrode, glucose 6-phosphate and ADP are produced from glucose and ATP by hexokinase, and the change in the amount of glucose 6-phosphate by the production thereof is converted into electric signals.
On the other hand, pyruvate kinase catalyzes the reproduction of ATP from the produced ADP and, then, the reproduced ATP is subjected to the above-mentioned enzyme reaction by hexokinase.
The repetition of these two reactions leads to an increase in the amount of glucose 6-phosphate produced by the enzyme reaction, which increase is proportional to the amount of ATP in an analyzed sample. As a result, the sensitivity of the enzyme electrode is enhanced so as to enable a quantitative analysis of a small amount of ATP which has conventionally been undetectable. With respect to the determination of ATP by using this enzyme electrode, it has been reported that the measuring sensitivity to ATP is approximately 10−9 M.
However, this enzyme electrode has the following problems.
Hexokinase used in this enzyme electrode is an allosteric enzyme and the enzyme reaction rate of such an enzyme is difficult to control because the enzyme activity is easily influenced by substances other than its substrate. Therefore, a quantitative analysis of ATP has been difficult, especially when the ATP concentration of the sample is low.
Further, in this enzyme electrode, the enzymes are immobilized by an inclusion method which uses a gelatin membrane. When such an enzyme electrode is used for the analysis, in order to prepare a uniform sample solution and to facilitate the penetration of the sample solution into the gelatin membrane containing the enzymes, it is necessary to produce a flow of the sample solution by means of an agitator or stirrer. As a consequence, miniaturization of an apparatus employing the above-mentioned enzyme electrode has been difficult.
As mentioned above, an enzymatic cycling of ATP/ADP system is conducted in this enzyme electrode. Since both of ADP and ATP are involved in this enzymatic cycling, the analysis performed using this enzyme electrode is influenced by the concentration of ADP in the sample. Therefore, in order to selectively determine the ATP concentration of a sample containing both of ATP and ADP, it is necessary to conduct a cumbersome pretreatment for removing ADP from the sample.
As apparent from the above, there has been no conventional enzyme electrode which can be used for easily and rapidly performing a highly sensitive determination of the concentration of ATP in a sample.